Nitrogen - How does Nitrogen Enter the Biomass, and form Amino Acids Overview of the flow of nitrogen in the biosphere. Nitrogen, nitrites and nitrates are acted upon by bacteria (nitrogen fixation) and plants and we assimilate these compounds as in our diets. incorporation in animals occurs through the actions of and glutamine synthetase. Glutamate plays the central role in mammalian nitrogen flow, serving as both a nitrogen donor and nitrogen acceptor. Reduced nitrogen enters the human body as dietary free amino acids, protein, and the ammonia produced by intestinal tract bacteria. A pair of principal , glutamate dehydrogenase and glutamine synthetase, are found in all organisms and effect the conversion of ammonia into the amino acids glutamate and glutamine, respectively. Amino and amide groups from these 2 substances are freely transferred to other carbon skeletons by transamination and transamidation reactions. The nitrogen cycle. The total amount of nitrogen fixed annually in the biosphere exceeds 1011 kg. Reactions with red arrows occur largely or entirely in anaerobic environments. The redox states of the various nitrogen species are depicted at the bottom of the figure. N2 triple bond contains 945 kJ/mol of bond energy, vs. 351 kJ/mol for CO single bond. The organisms that do this are diazatrophs like Rhizobium. It is carried out by a unique system, Nitrogenase. It is a complex of two , Fe-protein, a homodimer that has a [4Fe-4S] cluster and two ATP binding sites. The other is the MoFe protein, a heterotetramer (α2β2). Each forms an αβ dimer that associates by 2 fold symmetry. Each dimer has two bound redox centers: (1) a P-cluster which consists of 2 [4Fe-3S] clusters linked an additional FeS. (2) Fe-Mo- which consists of a [4Fe-3S] cluster & a [Mo-3Fe-3S] cluster bridged by 3 S ions. Nitrogen fixation can be represented by the following equation, in which two moles of ammonia are produced from one mole of nitrogen gas, at the expense of 16 moles of ATP and a supply of electrons and protons: + - N2 + 8H + 8e + 16 ATP → 2NH3 + H2 + 16ADP + 16 Pi. This reaction is performed exclusively by prokaryotes, by the enzyme complex nitrogenase.

The reactions occur while N2 is bound to the nitrogenase enzyme complex. The Fe-protein is first reduced by electrons donated by ferredoxin. Then the reduced Fe protein binds ATP and reduces the molybdenum-iron protein, which donates electrons to

N2, producing HN=NH. In two further cycles of this process (each requiring electrons donated by ferredoxin) HN=NH is reduced to H2N-NH2, and this in turn is reduced to 2NH3.

Depending on the type of microorganism, the reduced ferredoxin which supplies electrons for this process is generated by , respiration, or .! Nitrogen fixation by the nitrogenase complex

• Reduction of nitrogen to ammonia is exergonic, but costly in terms of amount of ATP required:

• The nitrogen triple bond is very stable, with a bond energy of 945 kJ/mol.

• The high activation energy is partially overcome by binding and hydrolysis of ATP. The overall reaction is:

6 Role of ATP in nitrogen fixation

• The binding of ATP to dinitrogenase reductase, and the subsequent hydrolysis of ATP, result in conformational changes that help to overcome the high activation energy of nitrogen fixation.

• Specifically, ATP binding results in lowering the reduction potential (E’°) of the reductase from – 300 mV to –420 mV, which enhances its reducing power.

Remember, electrons tend to flow spontaneously from carriers of lower E’° to carriers of higher E’° (think of oxidative phosphorylation). 7 + Glutamine and Glutamate as key entry points for NH4 Glutamine synthetase + enables toxic NH4 to combine with glutamate to yield glutamine.

Transamination reactions collect the amino groups from many different amino acids in the form of Glutamine synthetase L-glutamate. is found in ALL organisms. Ammonia transport in the form of glutamine. Excess ammonia in tissues is added to glutamate to form glutamine, a process catalyzed by glutamine synthetase. After transport in the bloodstream, the glutamine enters the + and NH4 is liberated in mitochondria by the enzyme glutaminase + Glutamine and Glutamate as key entry points for NH4 • Bacteria and plants have glutamate synthase

• Glutamate dehydrogenase also provides glutamate

Glutamate releases an amino group + as NH4 in the liver using glutamate dehydrogenase. • This enzyme occurs only in microorganisms, plants and lower animals. • Ammonia stored in glutamine is transferred to α-KG to form 2 glutamate. The reduction power comes from NADPH or ferredoxin. • The NADPH-dependent glutamate synthase from Azospirillum brazilense, is a heterotetramer, with α2 & β2 subunits. • The α subunit FMN and has a [4Fe-3S] cluster on each. • The β subunit has an FAD site and 2 [4Fe-4S] clusters. • The rxn is 5 steps that occurs in 3 active sites. [1] The electrons are transferred from NADPH to FAD at active site 1 on the β subunit.

[2] Electrons travel from the FADH2 to FMN at site 2 on the α subunit to yield FMNH2

[3] Glutamine is hydrolysed at site 3 to glutamate and NH3

[4] the NH3 moves thru the channel to site 2, where it reacts with α-KG .

[5] α-iminoglutarate is reduced by FMNH2 to form glutamate. Glutamate Synthase • glutamate synthase (NADPH) is an enzyme that catalyzes the chemical reaction • L-glutamine + 2-oxoglutarate + NADPH + H + 2 L-glutamate + NADP+ • Thus, the four substrates of this enzyme are L-glutamine, 2-oxoglutarate (α-ketoglutarate), NADPH, and H+ whereas the two products are L- glutamate and NADP+. • This enzyme belongs to the family of oxidoreductases, specifically those acting on the CH-NH2 group of donors with NAD+ or NADP+ as acceptor. This enzyme participates in glutamate metabolism and nitrogen metabolism. It has 5 cofactors: FAD, Iron, FMN, Sulfur, and Iron-sulfur. • It occurs in bacteria and plants but not animals, and is important as it provides glutamate for the glutamine synthetase reaction. • glutamate synthase (NADH); 2 L-glutamate + NAD+ L-glutamine + 2-oxoglutarate + NADH + H+

13 glutamate synthase (ferredoxin) (EC 1.4.7.1) is an enzyme that catalyzes the chemical reaction

2 L-glutamate + 2 oxidized ferredoxin L-glutamine + 2-oxoglutarate + 2 reduced ferredoxin + 2 H+

Thus, the two substrates of this enzyme are L-glutamate and oxidized ferredoxin, whereas its 4 products are L- glutamine, 2-oxoglutarate, reduced ferredoxin, and H+.

This enzyme participates in nitrogen metabolism. It has 5 cofactors: FAD, iron, sulfur, iron-sulfur, and flavoprotein.

14 Ammonia transport in the form of glutamine. Excess ammonia in tissues is added to glutamate to form glutamine, a process catalyzed by glutamine synthetase. After transport in the bloodstream, the glutamine enters the liver + and NH4 is liberated in mitochondria by the enzyme glutaminase The large size (MW ca. 620 Kda) and the complex regulation patterns of Glutamine Synthetase (GS) stem from its central role in cellular nitrogen metabolism. It brings nitrogen into metabolism by condensing ammonia with glutamate, with the aid of ATP, to yield glutamine. GS is from S.typhimurium, has Mn+2 bound, and is fully unadenylylated. Feedback Inhibition: Bacterial GS was previously shown to be inhibited by nine endproducts of glutamine metabolism. Each feedback inhibitor were proposed to have a separate site. However, x-ray data show: 1. AMP binds at the ATP substrate site. 2. The inhibiting amino acids Gly, Ala, and Ser bind at the Glu site. 3. Carbamyl-l- binds overlapping both the Glu and Pi sites. 4. The proximity of carbamyl- phosphate to the amino acid inhibitors hinders their binding to GS. Glutamine Synthetase can be composed of 8, 10, or 12 identical subunits separated into two face-to-face rings. Bacterial GS are dodecamers with 12 active sites between each monomer. Each active site creates a ‘bifunnel’ which is the site of three distinct substrate binding sites: , ion, and amino acid. ATP binds to the top of the bifunnel that opens to the external surface of GS. Glutamate binds at the bottom of the active site. The middle of the bifunnel contains two sites in which divalent cations bind (Mn+2 or Mg+2). One cation binding site is involved in phosphoryl transfer of ATP to glutamate, while the second stabilizes active GS and helps with the binding of glutamate.

Hydrogen bonding and hydrophobic interactions hold the two rings of GS together. Each subunit possesses a C-terminus and an N-terminus in its sequence. The C-terminus (helical thong) stabilizes the GS structure by inserting into the hydrophobic region of the subunit across in the other ring. The N-terminus is exposed to the solvent. In addition, the central channel is formed via six four-stranded β-sheets composed of anti-parallel loops from the twelve subunits. Cumulative feedback regulation of glutamine synthetase Cascade leading to adenylylation (inactivation) of glutamine synthetase.

GS is finely regulated by reversible inactivation involving a glutamate-dependent covalent attachment of an adenylyl group to a tyrosyl residue of each 12 subunits. This is catalyzed by an Adenylyltransferase (AT). It catalyses both the adenylation and denadenylation reactions. The adenylation to the 12 indentical subunits does not have to be total and the activity is dependent upon the degree of adenylation. The partially adenylated GS is more sensitive to feedback inhibition than the unadenylated enzyme. The degree of adenylylation is dependent upon over 40 metabolites. AT is a single peptide, 115kD. It is activated by ATP, glutamine and the PII regulatory protein. The activator of deadenylylation is αKG. PII regulatory protein can exist in two forms, uridylylate PII which stimulates deadenylylation and deuridylylated PII which stimulates adenylylation. This is catalyzed by Uridylyltransferase (UT). 20 The glutamine synthetase reaction is important in several respects. It produces glutamine. In animals, glutamine is the major amino acid found in the . Its role there is to carry ammonia to and from various tissues but principally from peripheral tissues to the , where the amide nitrogen is hydrolyzed by the enzyme glutaminase regenerates glutamate and free ammonium ion, which is excreted in the . S is present predominantly in the brain, kidneys, and liver. GS in the brain participates in the metabolic regulation of glutamate, the detoxification of brain ammonia, the assimilation of ammonia, recyclization of , and termination of signals. GS, in the brain, is found primarily in astrocytes. ] Astrocytes protect neurons against excitotoxicity by taking up excess ammonia and glutamate. Ammonia arising in peripheral tissue is carried in a nonionizable form which has none of the neurotoxic or alkalosis-generating properties of free ammonia. Liver contains both glutamine synthetase and glutaminase but the enzymes are localized in different cellular segments. This ensures that the liver is neither a net producer nor consumer of glutamine. The differences in cellular location of these two enzymes allows the liver to scavenge ammonia that has not been incorporated into . The enzymes of the urea cycle are located in the same cells as those that contain glutaminase. The result of the differential distribution of these two hepatic enzymes makes it possible to control ammonia incorporation into either urea or glutamine, the latter leads to excretion of ammonia by the kidney. 22 Enzyme-catalyzed transaminations. In many aminotransferase reactions, α-ketoglutarate is the amino group acceptor. All aminotransferases have pyridoxal phosphate (PLP) as cofactor. Although the reaction is shown here in the direction of transfer of the amino group to α- ketoglutarate, it is readily reversible Aminotransferases exist for all amino acids except and . The most common compounds involved as a donor/acceptor pair in transamination reactions are glutamate and a-ketoglutarate (a- KG), which participate in reactions with many different aminotransferases. equilibrate amino groups among available a-keto acids. This permits synthesis of non-essential amino acids, using amino groups derived from other amino acids and carbon skeletons synthesized in the cell. Thus a balance of different amino acids is maintained, as proteins of varied amino acid contents are synthesized. Although the amino N of one amino acid can be used to synthesize another amino acid, nitrogen must be obtained in the diet as amino acids Serum aminotransferases such as serum glutamate-oxaloacetate-aminotransferase (SGOT) (also called aspartate aminotransferase, AST) and serum glutamate-pyruvate aminotransferase (SGPT) (also called , ALT) have been used as clinical markers of tissue damage, with increasing serum levels indicating an increased extent of damage. has an important function in the delivery of skeletal muscle carbon and nitrogen (in the form of alanine) to the liver. In skeletal muscle, pyruvate is transaminated to alanine, thus affording an additional route of nitrogen transport from muscle to liver. In the liver alanine transaminase trasnfers the ammonia to a-KG and regenerates pyruvate. The pyruvate can then be diverted into . This process is referred to as the glucose-alanine cycle. Pyridoxal phosphate, the prosthetic group of aminotransferases. (b) Pyridoxal phosphate is bound to the enzyme through noncovalent interactions and a Schiff-base (aldimine) linkage to a Lys residue at the active site. The steps in the formation of a Schiff base from a primary amine and a carbonyl group Pyridoxal phosphate, the prosthetic group of aminotransferases. (c) PLP (red) bound to one of the two active sites of the dimeric enzyme aspartate aminotransferase, a typical aminotransferase 27 Glucose-alanine cycle. Alanine serves as a carrier of ammonia and of the carbon skeleton of pyruvate from skeletal muscle to liver. The ammonia is excreted as urea and the pyruvate is used to produce glucose, which is returned to the muscle.! Overview of amino acid biosynthesis

All amino acids are derived from intermediates in: • • the cyle • pentose phosphate pathway

Nitrogen enters these pathways by way of glutamate and glutamine. Transaminases equilibrate amino groups among available a-keto acids. This permits synthesis of non-essential amino acids, using amino groups derived from other amino acids and carbon skeletons synthesized in the cell. Thus a balance of different amino acids is maintained, as proteins of varied amino acid contents are synthesized. Although the amino N of one amino acid can be used to synthesize another amino acid, nitrogen must be obtained in the diet as amino acids

Summary of amino acid catabolism. Amino acids are grouped according to their major degradative end product. Some amino acids are listed more than once because different parts of their carbon skeletons are degraded to different end products. The figure shows the most important catabolic pathways in vertebrates, but there are minor variations among vertebrate species. Threonine, for instance, is degraded via at least two different pathways, and the importance of a given pathway can vary with the organism and its metabolic conditions. The glucogenic and ketogenic amino acids are also delineated in the figure, by color shading. Notice that five of the amino acids are both glucogenic and ketogenic. The amino acids degraded to pyruvate are also potentially ketogenic. Only two amino acids, and lysine, are exclusively ketogenic.

32 Glucogenic amino acids give rise to a net production of pyruvate or TCA cycle intermediates (i.e. alpha-ketoglutarate or oxaloacetate) that are precursors to glucose via gluconeogenesis.

All amino acids except lysine and leucine are at least partly glucogenic. Lysine and leucine are the only amino acids that are solely ketogenic, giving rise only to acetyl-CoA or Acetoacetyl-CoA, neither of which can bring about net glucose production.

A small group of amino acids comprised of , threonine, , , and give rise to both glucose and fatty acid precursors and are both glucogenic and ketogenic.

Finally, it should be recognized that amino acids have a third possible fate. During times of starvation the reduced carbon skeleton is used for energy production, with the result that it is oxidized to CO2 and H2O. Links between the urea cycle and . The interconnected cycles have been called the "Krebs bicycle." The pathways linking the citric acid and urea cycles are known as the aspartate-argininosuccinate shunt; these effectively link the fates of the amino groups and the carbon skeletons of amino acids. The interconnections are even more elaborate than the arrows suggest. For example, some citric acid cycle enzymes, such as and , have both cytosolic and mitochondrial isozymes. Fumarate produced in the — whether by the urea cycle, purine biosynthesis, or other processes—can be converted to cytosolic malate, which is used in the cytosol or transported into mitochondria (via the malate-aspartate shuttle) to enter the citric acid cycle. The glutamate dehydrogenase utilizes both nicotinamide nucleotide cofactors; NAD+ in the direction of nitrogen liberation and NADP+ for nitrogen incorporation. In the forward reaction as shown above glutamate dehydrogenase is important in converting free ammonia and alpha-ketoglutarate (a-KG) to glutamate, forming one of the 20 amino acids required for protein synthesis. However, it should be recognized that the reverse reaction is a key anapleurotic process linking amino acid metabolism with TCA cycle activity. In the reverse reaction, glutamate dehydrogenase provides an oxidizable carbon source used for the production of energy as well as a reduced electron carrier, NADH. As expected for a branch point enzyme with an important link to energy metabolism, glutamate dehydrogenase is regulated by the cell energy charge. ATP and GTP are positive allosteric effectors of the formation of glutamate, whereas ADP and GDP are positive allosteric effectors of the reverse reaction. Thus, when the level of ATP is high, conversion of glutamate to a-KG and other TCA cycle intermediates is limited; when the cellular energy charge is low, glutamate is converted to ammonia and oxidizable TCA cycle intermediates. The multiple roles of glutamate in nitrogen balance make it a gateway between free ammonia and the amino groups of most amino acids. The equilibrium position of Gdh favors the synthesis of glutamate, but studies show that in vivo it has an ΔG0´=0. All tissues have some capability for synthesis of the non-essential amino acids, amino acid remodeling, and conversion of non-amino acid carbon skeletons into amino acids and other derivatives that contain nitrogen.

However, the liver is the major site of nitrogen metabolism in the body. In times of dietary surplus, the potentially toxic nitrogen of amino acids is eliminated via transaminations, , and urea formation; the carbon skeletons are generally conserved as , via gluconeogenesis, or as fatty acid via pathways. In this respect amino acids fall into three categories: glucogenic, ketogenic, or glucogenic and ketogenic.

This shows the link between amino acid utilization as keto acids for the TCA cycle, transamination reactions and the Urea cycle. !

CPS is technically not part of the Urea cycle. It forms carbamyl PO4 which is one of the substrates of the transcarbamylase.

Eukaryotes have two forms of the enzyme, CPSI is found in the mitochondria matrix, CPSII is cytosolic and part of pyrimidine synthesis. CPSI uses NH3 while CPSII used glutamine as the Nitrogen donor.

The CPSI rxn is non-reversible and is the allosteric and rate limiting step of the Urea Cycle.

E.coli has only one CPS which is homologous to both enzymes found in eukaryotes. The enzyme exhibits substrate tunneling. The three sites of the rxn are found along a 96A X-Ray structure of E. coli synthetase tunnel in the elongate protein. (CPS).! Nitrogen-acquiring reactions in the synthesis of urea Nitrogen- acquiring reactions in the synthesis of urea. The urea nitrogens are acquired in two reactions, each requiring ATP. (b) In the reaction catalyzed by argininosuccinate synthetase, the second nitrogen enters from aspartate. Activation of the ureido oxygen of in step 1 sets up the addition of aspartate in step 2. Nitrogen-acquiring reactions in the synthesis of urea Synthesis of N- acetylglutamate and its activation of carbamoyl phosphate synthetase I. In the presence of excess glutamamte and acetyl-CoA there is the increased synthesis of N-acetyl- glumate, which is the the positive modulator aof CPS 1 activity. Links between the urea cycle and citric acid cycle. The interconnected cycles have been called the "Krebs bicycle." The pathways linking the citric acid and urea cycles are known as the aspartate-argininosuccinate shunt; these effectively link the fates of the amino groups and the carbon skeletons of amino acids. The interconnections are even more elaborate than the arrows suggest. For example, some citric acid cycle enzymes, such as fumarase and malate dehydrogenase, have both cytosolic and mitochondrial isozymes. Fumarate produced in the cytosol—whether by the urea cycle, purine biosynthesis, or other processes—can be converted to cytosolic malate, which is used in the cytosol or transported into mitochondria (via the malateaspartate shuttle; see Figure 19-29) to enter the citric acid cycle.